Agents for inhibition of chemoattractant

ABSTRACT

A chemoattractant protein called &#34;eotaxin&#34; is capable of attracting eosinophils and of inducing eosinophil accumulation and/or activation in vitro and in vivo. Various types of agents that inhibit or otherwise hinder the production, release or activity of eotaxin may be used therapeutically in the treatment of asthma and other inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATION

This is a 371 of WO 95/07985.

The present invention relates to a chemotactic cytokine. Theaccumulation of eosinophil leukocytes is a characteristic feature ofIgE-mediated allergic reactions such as allergic asthma, rhinitis andeczema. Eosinophil accumulation also occurs in non-allergic asthma. Theimmediate broncho-constriction in response to a provoking stimulus inthe asthmatic involves mast cell activation and the release ofconstrictor mediators. This is followed after several hours in someindividuals by a late bronchoconstrictor response associated with amassive influx of eosinophils (1). Repeated provocation results inchronic inflammation in the airways and a marked hyper-responsiveness toconstrictor mediators. The magnitude of both the late response and thechronic hyper-responsiveness correlates with the numbers of eosinophilspresent in the lung (2,3).

The present invention provides a chemoattractant protein capable ofattracting eosinophils and of inducing eosinophil accumulation and/oractivation in vitro and in vivo. The chemoattractant protein of thepresent invention is designated "eotaxin".

Eotaxins are proteins of the C--C branch of the platelet factor 4superfamily of chemotactic cytokines. Within the C--C branch of theplatelet factor 4 superfamily of chemotactic cytokines, or chemokines,certain members have the property of attracting eosinophils in vitro andsome may induce eosinophil accumulation in vivo. For example, thechemokines RANTES and MIP-1α attract eosinophils in vitro while MCP-1and MIP-1β do not. ("RANTES" denotes Regulated upon Activation in NormalT cells Expressed and Secreted, "MIP" denotes Macrophage InflammatoryProtein, and "MCP" denotes Monocyte Chemo-attractant Protein.)

Naturally-occurring cytokines within the platelet factor 4 superfamilyof chemotactic cytokines may have marked inter--the amino acid sequenceof the protein, and in the carbohydrate modifications of the protein,while retaining the same characteristic functional properties. Similarvariations in structure may occur in cytokines obtained from differentindividuals within the same species. Many chemokines within the C--Cbranch of the platelet factor 4 superfamily show promiscuity of receptorbinding, and the ability of different chemokines to bind to the samereceptor is not necessarily dependent on a high degree of homology atthe amino acid level. Accordingly, both interspecies and intraspeciesvariations in protein length, amino acid sequence and carbohydratemodifications are generally to be expected for eotaxins.

The ability to attract eosinophils and to induce eosinophil accumulationand/or activation in vitro and in vivo is a characteristic property ofeotaxins. Furthermore, eotaxins generally show substantially noattractive effect for neutrophils in vivo. The eosinophilchemoattractant effect may be an inter-species effect, for example,guinea-pig eotaxin appears to be potent in inducing chemotaxis of humaneosinophils in vitro.

An eotaxin may be obtained from an appropriate body fluid, for example,from bronchoalveolar lavage fluid obtained from a human or non-humansubject, particularly an allergic subject after an allergen challenge,either experimentally induced or naturally incurred. Other sources ofeotaxins are, for example, inflammatory exudate fluids and in vitrocultures of macrophages, lymphocytes, neutrophils, mast cells, airwayepithelial cells, connective tissue cells, vascular endothelial cellsand eosinophils themselves.

For example, an eotaxin may be obtained from a sensitised guinea-pigafter allergen challenge. Guinea-pig models are useful as they sharemany common features with the asthmatic response in man. Eotaxinobtainable from bronchoalveolar lavage fluid of a sensitised guinea-pigby sequential HPLC purification generally has a molecular weight in therange of from 6-16 kDa. (As indicated above, intraspecies molecularweight variations of this order of magnitude are observed in members ofthe platelet factor 4 superfamily.)

The amino acid sequence of a guinea-pig eotaxin is set out in SEQ.ID.NO. 1, SEQ.ID. NO. 2 and in FIGS. 7 and 8 of the accompanying drawings.Other guinea-pig eotaxins will generally have at least 50% overallhomology with the sequence shown in SEQ.ID. NO. 1 (FIG. 7) at the aminoacid level. The homology may be at least 60%, for example at least 70%,for example at least 80% with the sequence set out in SEQ.ID. NO. 1 andin FIG. 7.

Percentage homology in the present case is calculated on the basis ofamino acids that are identical in corresponding positions in the twosequences under investigation. Conservative substitutions are not takeninto account. In the calculation of percentage homology of a putativeeotaxin molecule under investigation with the sequence shown in SEQ.ID.NO. 1 (FIG. 7) or with SEQ.ID. NO. 2 (FIG. 8) if the molecule underinvestigation has a different length from the eotaxin set out in SEQ.ID.NO. 1 or SEQ.ID. NO. 2 (FIG. 7 or FIG. 8), then the calculation is basedon the amino acids in the portion of the molecule under investigationthat overlaps with the sequence shown in SEQ.ID. NO. 1 (FIG. 7) orSEQ.ID. NO. 2 (FIG. 8). Software packages for the alignment of aminoacid sequences and the calculation of homology are availablecommercially, for example, the "Bestfit" program available from GeneticsComputer Group Sequence Analysis Software, Madison, Wis., U.S.A.

Unless specified otherwise, the specific values of percentage homologybetween eotaxin and other chemotactic cytokines given in the presentspecification have been calculated on the basis of the eotaxin set outin SEQ.ID. NO. 1 (FIG. 7).

As indicated above, eotaxins obtainable from species other thanguinea-pigs, for example humans, will exhibit inter-species differencesof the type demonstrated by other members of the C--C branch of theplatelet factor 4 superfamily of chemokines, for example, differences inprotein length, amino acid sequence and carbohydrate modifications.There may, for example, be variations in the C- and/or N-terminalresidues.

For example, it is expected that the molecular weight of an eotaxin froma species other than guinea-pig will generally fall within the range offrom 6 kDa to 16 kDa, but in some cases an eotaxin may have a molecularweight less than 6 kDa or more than 16 kDa.

Similarly, it is expected that in general an eotaxin from a speciesother than guinea-pig will have at least 40% overall homology with thesequence set out in SEQ.ID. NO. 1 and in FIG. 7. The homology may be atleast 50%, for example at least 60%, for example at least 70%, forexample at least 80% with the sequence set out in SEQ.ID. NO. 1 and inFIG. 7. There may, however, be eotaxins from species other thanguinea-pigs that have less than 40% homology with SEQ.ID. NO. 1 (FIG.7).

Eotaxins may be identified by any one or more of the characteristics setout above, in particular by their ability to attract and/or activeeosinophils in vitro and cause their accumulation and/or activation invivo. A characteristic that assists the identification of a molecule asan eotaxin is the lack of attractive effect on neutrophils.

The present invention provides a method of determining the ability of asubstance to induce eosinophil accumulation and/or activation in vivo,that is to say, a method for testing putative eotaxins, which comprisesadministering the substance, generally intradermally, to a test animalpreviously treated with labelled, for example ¹¹¹ In-labelled,eosinophils and subsequently determining the number of labelledeosinophils at a skin site.

One in vitro method that may be used to test a putative eotaxin for theability to attract and/or activate eosinophils in vitro is the abilityof the substance to increase eosinophil intracellular calcium levels.Other general methods for determining chemotactic activity in vitro maybe used to test putative eotaxins in vitro.

Confirmation that an eosinophil attractant is an eotaxin may also bemade by consideration of sequence homology of that protein with thesequence set out in SEQ.ID. NO. 1 (FIG. 7) and/or with the sequence setin SEQ.ID. NO. 2 (FIG. 8) and/or by consideration of the structuralrelationship between the protein and the guinea-pig eotaxin.

As mentioned above, RANTES and MIP-1α are both eosinophil activators.Eotaxin has functional similarities but low structural homology withRANTES and MIP-1α (31% homology with MIP-1α at the amino acid levelcalculated on the basis of SEQ.ID. No. 1 (FIG. 7); 32% homology whencalculated on the basis of the overlapping sequences and 26% homologywith RANTES at the amino acid level calculated on the basis of SEQ.ID.No. 1 (FIG. 7); 27% homology when calculated on the basis of theoverlapping sequences). An eotaxin can be distinguished from RANTES andMIP-1α not only by the degree of homology but also by the overalldifferences in sequence and structure.

In addition to full-length eotaxin molecules, the present invention alsoprovides molecules that comprise less than a full length eotaxinsequence. Such molecules (called "fragments" herein) may be polypeptidesor peptides. For use as an eotaxin substitute, a fragment should retainone or more of the biological activities of the parent molecule.

Eosinophils contain an armoury of chemicals necessary for killingparasites. These chemicals have been implicated in the damage to airwayepithelium that occurs in asthma and may relate to the observed changesin airway function (26,27). From our studies we suggest that eotaxinsshould be considered as important mediators of eosinophil accumulationin vivo. Macrophages, lymphocytes, neutrophils, mast cells, airwayepithelial cells, connective tissue cells, vascular endothelial cellsand eosinophils themselves are likely candidates as the source of theeosinophil chemoattractant activity generated in the lung. Platelets mayalso have a role as it has been shown that they can release C--Cchemokines (22). Further, an early platelet deposition may be involvedin the subsequent eosinophil accumulation in vivo (28,29) and there isevidence that platelet-activating factor induces the synthesis of anunidentified eosinophil chemoattractant in vivo (30). In this respect,it is of interest that platelet-derived growth factor can induce geneexpression of C--C chemokines in fibroblasts (31). Furthermore, the C--Cchemokines have been implicated in wound healing (18). This may beimportant in the sub-epithelial basement membrane fibrosis that is aprominent feature of the asthmatic lung. Thus, eotaxins may be involvedin both eosinophil accumulation and in chronic structural changes in thelung.

Eotaxins may have an important role in asthma and in other diseaseshaving an inflammatory component where eosinophil accumulation and/oractivation is a prominent feature, for example, rhinitis and eczema,especially allergic eczema. Accordingly, agents that inhibit orotherwise hinder the production, release or action of eotaxins havepotential as selective therapeutic agents. Such agents and theirtherapeutic use are part of the present invention.

Such agents include inhibitors that affect the interaction of an eotaxinwith eotaxin receptors, for example, by binding to an eotaxin or to aneotaxin receptor. An example of such an inhibitor is receptorsthemselves which, on administration, can bind an eotaxin and prevent itsinteraction with naturally-occurring receptors. Such inhibitoryreceptors may be soluble or insoluble. Receptors which are not involvedin cell activation may be bound to, or induced on, cells. Such receptorsmay also be used to remove endogenous eotaxin.

Further examples of agents that affect the interaction of eotaxins witheotaxin receptors are receptor antagonists, and antibodies, bothantibodies directed against (capable of binding with) an eotaxin andantibodies directed against an eotaxin receptor, especially monoclonalantibodies. Any other agent that inhibits or otherwise hinders thebinding of an eotaxin to an eotaxin receptor also has therapeuticpotential, for example, any other agent that binds to an eotaxin or toan eotaxin receptor. Further agents that have therapeutic potential arethose that prevent or reduce activation of eotaxin receptors.

Further agents that inhibit or otherwise hinder the action of eotaxinsare those that change the structure of an eotaxin such that it is nolonger able to bind to an eotaxin receptor, for example, an enzyme orother agent that degrades eotaxin specifically.

Receptor promiscuity is common among chemokines, so although it isessential that a receptor is capable of binding an eotaxin, the receptorneed not necessarily be eotaxin-specific. For example, a receptor maybind MIP-1α, RANTES and/or other eosinophil attractant chemokines aswell as an eotaxin.

As indicated above, possibilities for therapeutic intervention includethe use of a receptor to which an eotaxin binds, especially a solublereceptor. It may be advantageous to use an eotaxin-specific receptor.Further possibilities for therapeutic intervention include receptorantagonists, for example, based on 3-dimensional structures or the aminoacid sequences of eotaxins and/or of eotaxin receptors, and agents foundto inhibit eotaxin or other agonists binding to or activating eotaxinreceptors. For example, a receptor antagonist or an agonist inhibitormay be a polypeptide in which the sequence of a full-lengthnaturally-occurring eotaxin has been modified, for example, by aminoacid substitution, or may be a fragment of an eotaxin (that is to say, apolypeptide or small peptide comprising part of the amino acid sequenceof a naturally-occurring eotaxin), or a modified fragment of an eotaxin,for example, modified by amino acid substitution.

Furthermore, knowledge of the sequence and/or structure of eotaxinseither alone or in combination with knowledge of the sequence and/orstructure of other chemokines that bind to the same receptor(s) aseotaxins, provides useful information for the design of therapeuticagents.

Agents that prevent or inhibit eotaxin synthesis or release may also beused therapeutically. Such agents and their use are also part of thepresent invention.

All inhibitors of eotaxin activity, synthesis and release, includingsoluble receptors, antibodies, antagonists and inhibitors of agonistbinding, and their use are part of the present invention.

The present invention accordingly provides an agent that inhibits orotherwise hinders the production, release or action of an eotaxin,especially an agent as described above, for use as a medicament. Theinvention also provides the use of an agent that inhibits or otherwisehinders the production, release or action of an eotaxin, especially anagent as described above, in the manufacture of a medicament for thetreatment of asthma or another disease having an inflammatory component,particularly with accumulation of eosinophils, for example, rhinitis oreczema, especially allergic eczema.

The use of the structural and sequence information relating to eotaxinsin the design of therapeutically and diagnostically useful agents, forexample, in computer-aided design based on the three dimensionalstructure of eotaxins is part of the present invention.

Putative inhibitors of eotaxin activity may be screened using in vivoand in vitro assays based on inhibition of chemoattraction and/oraccumulation and/or activation of eosinophils by eotaxins. Some generalmethods for testing the activity of a compound for an inhibitory effecton the activity of a chemoattractant cytokine in vitro are known. Suchassays may be used to determine the inhibitory action of a putativeinhibitor on in vitro effects induced in eosinophils by eotaxins.

Assays that are suitable for screening putative eotaxin inhibitorsinclude, for example, inhibition in vitro of elevation of intracellularcalcium levels induced in cells by eotaxin. The method of the presentinvention for determining the ability of a substance to induceeosinophil accumulation and/or activation in vivo, that is to say, amethod for testing putative eotaxins, may also be used to determine theability of a substance to inhibit eosinophil accumulation and/oractivation induced in vivo by an eotaxin: an animal is pretreated withlabelled eosinophils, an eotaxin and a putative inhibitor areadministered, and the number of labelled eosinophils at a skin site aredetermined subsequently. The eotaxin is generally administeredintradermally, and the putative inhibitor may be administered by thesame route or by a different route, for example systemically.

Examples of in vitro and in vivo assays both for the determination ofeotaxin activity and for the determination of eotaxin inhibitoryactivity are described herein. For example, Example 1 gives a detailedprotocol for the in vivo assay of the present invention, and Example 4gives detailed protocols of various assays. The assays described hereinmay be used as such, or may be modified as required. Assays may be usedalone or in combination to establish eotaxin and eotaxin-inhibitoryactivity. A putative inhibitors may be any of the types of moleculesdescribed above, including receptors, for example, soluble receptors,antibodies, and antagonists and inhibitors of agonist binding. Methodsfor testing putative inhibitors of eotaxins are also part of the presentinvention.

A further aspect of the present invention is a pharmaceuticalpreparation comprising, as active ingredient, an agent that inhibits orotherwise hinders the production, release or action of an eotaxin, inadmixture or in conjunction with a pharmaceutically suitable carrier.Such agents are described above and include, for example, an inhibitorof eotaxin synthesis or release, a soluble eotaxin receptor, an eotaxinreceptor antagonist or an inhibitor of an eotaxin receptor agonist, anantibody against eotaxin or an antibody against an eotaxin receptor.

The invention further provides a method of treating asthma and otherinflammatory diseases, comprising the administration of an effectiveamount of an agent that inhibits or otherwise hinders the production,release or action of an eotaxin. The agent may be as described above,for example, an inhibitor of eotaxin synthesis or release, a solubleeotaxin receptor, an eotaxin receptor antagonist or an inhibitor of aneotaxin receptor agonist, or an antibody against eotaxin or against aneotaxin receptor.

The present invention also provides assays for eotaxins and foranti-eotaxin antibodies, especially immunoassays and in particularELISAs (enzyme-linked immunosorbent assays). The invention provides, forexample, an immunoassay for an antigen, characterised in that theantigen is an eotaxin, and also provides an immunoassay for an antibody,characterised in that the antibody is an anti-eotaxin antibody. Theinvention also provides assays for eotaxins that are analogous toimmunoassays for eotaxins but that use a specific-binding partner otherthan an antibody. In such specific-binding partner assays an eotaxinreceptor may be used instead of an anti-eotaxin antibody.

In an immunoassay, an anti-eotaxin antibody may, for example, be coatedon a solid surface to enable capture and hence detection of eotaxin. Ananti-eotaxin antibody may be used in an assay for the detection ofantibodies to eotaxin, for example, in a competitive antibody assay. Alabelled eotaxin or a derivative thereof, for example, a recombinanteotaxin or a synthetic peptide comprising part of the amino acidsequence of an eotaxin may be used in a competitive antigen assay foreotaxin or may be used to coat a solid surface in a capture assay forantibodies to eotaxin. The many different types of assay format are welldescribed in the literature of the art, see for example "ELISA and OtherSolid Phase Immunoassays, Theoretical and Practical Aspects" Eds. KemenyD. M. & Challacombe S. J., John Wiley, 1988. (36). Assays using aneotaxin receptor instead of an anti-eotaxin antibody may be carried outanalogously.

The present invention provides a process for the production of aneotaxin, which comprises obtaining bronchoalveolar lavage fluid obtainedfrom a human or non-human animal challenged with a provoking stimulus,for example, from a human suffering from allergic or non-allergicasthma, or other lung disease, or a guinea-pig sensitised with a foreignprotein, and isolating a fraction showing eosinophil chemoattractantactivity. One method of isolating an eotaxin-containing fraction ofbronchoalveolar lavage fluid is sequential cation exchange, sizeexclusion and reversed phase HPLC systems. The desired fractiongenerally contains a polypeptide having a molecular weight in the rangefrom 6-6 kDa. Purity may be verified by SDS-PAGE. If desired, theauthenticity of the substance obtained may be determined by comparisonof the amino acid sequence thereof with the amino acid sequence set outin SEQ.ID. NO. 1 or SEQ.ID. NO. 2 (FIG. 7 or FIG. 8).

Eotaxins may be obtained according to the above procedure from othersources, for example, from inflammatory exudate fluids, or from in vitrocultures of macrophages, lymphocytes, neutrophils, mast cells, airwayepithelial cells, connective tissue cells, vascular endothelial cellsand eosinophils themselves.

Alternatively, a full-length eotaxin, or a part (fragment) of aneotaxin, for example, a polypeptide or peptide fragment, may be producedby chemical synthesis, for example, according to the Merryfieldtechnique. A further method for producing a full-length eotaxin or apart thereof is by recombinant DNA technology. All methods of producingeotaxin are part of the present invention.

To produce a full-length eotaxin polypeptide or a polypeptide (orpeptide) fragment by recombinant DNA technology, a nucleic acid sequenceencoding the polypeptide is inserted into an expression vector under thecontrol of appropriate control sequences. A recombinant polypeptide maythen be expressed using a prokaryotic expression system, for example, inE. coli, or using a eukaryotic cell system, in which case the resultingpolypeptide may be glycosylated. Such techniques are standard see, forexample Sambrook, J., Fritisch, E. F. and Maniatis T., Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989 (37).

A nucleic acid encoding all or part of an eotaxin polypeptide may beobtained by screening a library prepared from suitable cells, forexample, cells from allergen-challenged guinea-pig or human lung.Screening may be carried out using a probe comprising sequencescharacteristic of an eotaxin, in particular, sequences that distinguishthe eotaxin from other related cytokines, for example, RANTES andMIP-1α. It may be preferable to use a long probe, for example, a probecomprising a nucleic acid sequence encoding a full-length eotaxinpolypeptide.

Alternatively, the polypeptide sequence of an eotaxin may be used todesign oligonucleotide primers, for example, degenerate primers. Primersmay, for example, comprise bases less specific in their interaction thanthe naturally-occurring bases, for example, inosine may be used.Examples of primers are the sense sequence

5' TGC TGT TTC CGI GTI ACI AAC AAA (SEQ.ID.NO. 3)

based on the amino acid sequence CCFRVTNK, and the anti-sense sequence

5' CAT CTT GTC IGG CTT IAT TTC (SEQ.ID. NO. 4)

based on the amino acid sequence EIKPDKM. Such primers may be used foramplification of reverse transcribed mRNA by the polymerase chainreaction. This provides cDNA probes for screening libraries, forexample, as described above, to isolate full length eotaxin clones.Primers may include codons chosen on the basis of known speciespreference.

As indicated above, derivatives of naturally occurring eotaxins are alsopart of the present invention. Such derivatives include polypeptidesthat have one or more of the following modifications relative to anaturally-occurring eotaxin:

(i) elongation or shortening at the C-terminus;

(ii) elongation or shortening at the N-terminus;

(iii) deletion and/or insertion of internal sequences;

(iv) amino acid substitutions, for example at the C- and/or N-terminus;and

(v) a different pattern of glycosylation. (There is, for example, apotential O-glycosylation site at residue 70.)

Such derivatives may function as agonists for structure/activityrelationship studies or as receptor antagonists.

Anti-eotaxin antibodies and anti-eotaxin receptor-antibodies, bothpolyclonal and monoclonal, may be produced according to standardtechniques, for example, Kohler & Milstein (38). A full-length eotaxinmay be used as antigen, or it may be preferred to use a fragment of aneotaxin in order to produce an antibody to a specific antigenicdeterminant. A naturally occurring eotaxin may used as antigen.Alternatively, a recombinant or synthetic eotaxin or eotaxin polypeptideor peptide may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 18 of the accompanying drawings illustrate the presentinvention. The following is a brief description of the Figures. A moredetailed description of the Figures is given below and in the Examplessection of this specification.

FIG. 1: Protocol for the generation and assay of eosinophilchemoattractant activity in vivo.

FIG. 2: Time course of generation of eosinophil chemo-attractantactivity in lungs of sensitized guinea-pigs after antigen challenge.

FIGS. 3, 4 and 5 all relate to the purification of eotaxin frombronchoalveolar lavage (BAL) fluids:

FIG. 3: Final reversed phase HPLC profile showing absorbance at 214 nmand the acetonitrile gradient.

FIG. 4: Measurement of ¹¹¹ In-eosinophil chemoattractant activity of thefractions obtained on HPLC chromatography.

FIG. 5: Measurement of ¹¹¹ In-neutrophil chemo-attractant activity; lackof significant activity observed.

FIG. 6: SDS-PAGE analysis of C18 reversed phase HPLC fractions 50-56.

FIGS. 7 and 8: Amino acid sequences of an isolated guinea-pig eotaxin.

FIG. 9: Comparison of the eotaxin sequence of FIG. 7 with humanMCP-1(SEQ ID NO:5), MCP-2(SEQ ID NO:6), MCP-3 (21) (SEQ ID NO:7),guinea-pig MCP-1 (24) (SEQ ID NO:8), human MIP-1α (SEQ ID NO:9), MIP-1β(SEQ ID NO:10) and RANTES (SEQ ID NO:11) (18) showing conserved residues(shaded).

FIG. 10: Comparison of ¹¹¹ In-eosinophil accumulation in vivo usingguinea-pig eotaxin and the recombinant human proteins RANTES, MIP-1α andMCP-1.

FIG. 11 (Inset): Inhibition of the binding of ¹²⁵ I-RANTES to guinea-pigeosinophils in vitro induced by eotaxin and RANTES but not by a memberof the C-X-C branch of chemokines, IL-8.

FIGS. 12, 13 and 14 illustrate the potential of blocking the response toguinea-pig eotaxin. This makes use of the fact that human RANTES, whilstcompeting with eotaxin for binding sites, does not activate guinea-pigeosinophils.

FIG. 12: Elevation of intracellular calcium levels in human eosinophilsin vitro induced by eotaxin, RANTES and, at high concentration only,MCP-1.

FIG. 13: Elevation of intracellular calcium levels in guinea-pigeosinophils in vitro induced by eotaxin, but not human RANTES and MCP-1.

FIG. 14: Representative traces showing inhibition of guinea-pigeotaxin-induced elevation of intracellular calcium levels in guinea-pigeosinophils in vitro by the previous exposure of the same cells to humanRANTES. Inset:

FIG. 15: Values (mean±SEM) for four separate eosinophil preparations.

FIG. 16: Dose response curve showing inhibition of guinea-pigeotaxin-induced elevation of intracellular calcium levels in guinea-pigeosinophils in vitro by the previous exposure of the same cells to humanRANTES.

FIG. 17: Histogram showing comparison of in viva response of guinea-pigeosinophils to eotaxin, RANTES and RANTES plus eotaxin co-injectedintradermally.

FIG. 18: Histogram showing number of eosinophils and neutrophils inbronchoalveolar lavage fluid (BAL) after administration of guinea-pigeotaxin as an aerosol to guinea-pig airways.

As in allergic asthmatic patients, exposure of sensitised guinea-pigs toaerosolised antigen results in an immediate phase of bronchoconstrictionwith associated mast cell degranulation followed, in some individuals,by a late phase of bronchoconstriction and airway hyper-responsiveness(4-7). Although clearly no one model mimics all the features of thehuman disease, the guinea-pig model shares common features with theasthmatic response in man and has been extensively used to investigatepossible mechanisms (7). In particular, in both guinea-pig and man, theimmediate response to allergen triggers the subsequent accumulation inthe lung of high numbers of eosinophils. Experiments were designed todetect the appearance in the lung of chemoattractants that may beresponsible for the accumulation of eosinophils. A strategy was employedthat has previously been applied to the identification of neutrophilchemoattractants in inflammatory exudates (8-10).

Guinea-pigs were sensitised with intraperitoneal ovalbumin on day 1followed by a short exposure of their lungs to aerosolised ovalbumin atday 8. On day 15-21 animals were challenged with aerosolised ovalbuminand killed at different intervals using a barbiturate overdose.Immediately after death the airways were lavaged with saline. Thebroncho-alveolar lavage (BAL) fluid was centrifuged to remove cells, andsupernatants were either stored at -20° C. for assay or subjected topurification. Eosinophil chemoattractant activity in BAL fluid samplesor HPLC fractions was tested by injecting them intradermally into assayguinea-pigs previously given intravenous injections of ¹¹¹In-eosinophils (11,12). After a 2 or 4 h interval, assay animals werekilled and the punched out skin sites were counted in a gamma-counter(FIG. 1).

FIG. 2 shows the time-course of appearance of eosinophil chemoattractantactivity in BAL fluid. Significant activity was observed 30 min afterantigen challenge. Activity increased up to 3 h, remained high at 6 h,but was not significant in 24 h samples. Control samples (BAL fluid fromsham-sensitised/challenged or sensitised/sham-challenged guinea-pigs)taken at 3 h had no significant activity.

Eosinophil chemoattractant activity, which we termed eotaxin, waspurified from 3 h BAL fluid by sequential cation exchange, sizeexclusion and reversed phase HPLC using the in vivo ¹¹¹ In-eosinophilaccumulation assay to measure the activity of fractions throughout.Eotaxin eluted as a single discreet peak of bioactivity from both thecation exchange and the size exclusion steps, indicating a stronglycationic protein of 7-14kDa. Reversed phase chromatography separatedeosinophil chemoattractant activity into two peaks (fractions 51+52 andfraction 54), which were associated with discreet peaks of proteinabsorbance (FIGS. 3 and 4). Selectivity for eosinophils was shown by thelack of significant neutrophil chemoattractant activity in thesefractions as measured by the accumulation of ¹¹¹ In-neutrophils in theskin assay (FIG. 5). Histological examination of skin injected witheotaxin (2 pmol) demonstrated eosinophil accumulation at 4 and 24 h,particularly around small blood vessels (haematoxylin and eosin stains).

SDS-PAGE analysis revealed a single protein band in each of fractions51, 52 and 54 (FIG. 6). The protein in fractions 51 and 52 was slightlylarger than that in fraction 54. This was confirmed by mass analysis inwhich the major signals were at approximately 8.81 and 8.38 kDarespectively (see Example 3). N-terminal sequencing of fractions 51, 52and 54 revealed identical amino acid sequences (see FIG. 7). TheN-terminal 37 residue sequence of eotaxin as set out in SEQ.ID. NO. 1and FIG. 7 shows 57% homology with human monocyte chemotactic protein(MCP-1 (13), otherwise known as MCAF (14) and JE (15)). Tryptic peptidesof fraction 54 were also sequenced and readily aligned by comparisonwith human MCP-1 to give the virtually complete sequence of eotaxin withan overall homology of 53%, see FIG. 7. It is likely that the variationsin molecular mass reflect differential glycosylation as the four masssignals obtained (two major and two minor, see Example 3) are alldifferent from each other by multiples of approximately 220 mass units.The sequence contains no N-glycosylation sites, but a potentialO-glycosylation site at position 70 has been identified (see Example 3).Human MCP-1 also exhibits heterogeneity on SDS-PAGE due to differencesin the O-linked carbohydrate modification (16).

The platelet factor 4 superfamily of chemotactic cytokines, orchemokines, is characterised by four conserved cysteines. The relativeposition of the two N-terminal cysteines allows the subdivision of thissuperfamily into the C-X-C chemokines (eg. IL-8 (17)) which arepredominantly neutrophil chemoattractants and the C--C chemokines (eg.MCP-1, RANTES, MIP-1α and MIP-1β (18)) which are chemotactic forleukocytes other than neutrophils. Eotaxin is a member of the C--Cbranch of chemokines. The greatest homology (53%) is with human MCP-1which, in the limited in vitro studies to date, has been reported to beinactive on human eosinophils (19,20) and with the recently describedhuman MCP-2 (54%) and MCP-3 (51% homology calculated on the basis ofSEQ.ID. No. 1 (FIG. 7); 54% homology when calculated on the basis of theoverlapping sequences) (21). Homology with other human C--C chemokines(FIG. 9) is: MIP-1β (37% calculated on the basis of SEQ.ID. No. 1 (FIG.7); 39% when calculated on the basis of the overlapping sequences),MIP-1α (31% calculated on the basis of SEQ.ID. No. 1 (FIG. 7); 32% whencalculated on the basis of the over-lapping sequences) and RANTES (26%calculated on the basis of SEQ.ID. No. 1 (FIG. 7); 27% when calculatedon the basis of the overlapping sequences). The latter two proteins haverecently been shown to be potent eosinophil activators in vitro (20,22)whereas MIP-1β activates lymphocytes in vitro (23) but apparently noteosinophils (20). Eotaxin shows the greatest structural homology withhuman MCP-1, MCP-2 and MCP-3. Eotaxin has functional similarities, butrelatively low homology, when compared with RANTES and MIP-1α. Eotaxinis clearly a distinct molecule from guinea-pig MCP-1; the latter hasrecently been cloned (24) and it has only a 43% homology with theeotaxin sequence, see FIG. 9. Guinea-pig MCP-1 was shown to bechemotactic for monocytes but was not tested on eosinophils (24).Interestingly, eotaxin has a 41% homology with a C--C protein whose geneis expressed in mouse mast cells and upregulated 2 h after theinteraction between IgE and antigen (25). No functional activity hasbeen reported for this protein but it is distinct (51% homology) frommouse MCP-1/JE (25).

Guinea-pig eotaxin was very potent in inducing eosinophil accumulationin vivo: 1-2 pmol/skin site giving a 730±140% response (mean±s.e.m.,n=18 animals) compared with saline-injected sites. Further, markedeosinophil accumulation was seen within 30 minutes of intradermalinjection (FIG. 10). This is supported by experiments in vitro. Theeffect of eotaxin on eosinophils in vitro was shown by (i) inhibition ofbinding of ¹²⁵ I-RANTES to guinea-pig cells (FIG. 11), (ii) elevation ofcytoplasmic calcium in human and guinea-pig cells (FIGS. 12 and 13) and(iii) chemotaxis of human eosinophils in a Boyden chamber system: thechemotactic responses to eotaxin and RANTES were of similar magnitudeover the range 0.1-3.0 nM. In contrast to eotaxin, human recombinantMCP-1 and RANTES did not induce guinea-pig eosinophil responses in vivoor in vitro (FIGS. 10 and 13). This may reflect a species differencealthough RANTES did bind to guinea-pig eosinophils without inducingactivation (FIG. 11, inset in FIG. 10). Eotaxin has a similar potency toRANTES on human eosinophils whereas MCP-1 is either inactive (20) oractive only at high doses (FIG. 12). Thus, eotaxin has a potent directeffect on both human and guinea-pig eosinophils.

Responses of guinea-pig eosinophils to guinea-pig eotaxin in vitro andin vivo can be inhibited by human RANTES. FIGS. 14, 15 and 16 show theresponse of FURA-2-loaded guinea-pig eosinophils to guinea-pig eotaxinbefore and after the addition of human RANTES. The eotaxin-inducedincrease in intracellular calcium concentration is reduced to asubstantial degree when the cells are first exposed to human RANTES,which itself fails to induce a response. FIG. 17 shows that humanRANTES, when coinjected with the eotaxin, reduces the accumulation ofguinea-pig eosinophils induced by eotaxin in guinea-pig skin in vivo.The results support our observations that eotaxin exhibits competitivebinding with radiolabelled human RANTES on guinea-pig eosinophils andthat eotaxin is a potent functional stimulant whereas RANTES is not.Accordingly, RANTES appears to act as a receptor antagonist for eotaxinon guinea-pig eosinophils.

FIG. 18 shows that eosinophils accumulate in guinea-pig airways in vivowithin 24 hours of the administration of aerosolized guinea-pig eotaxin,whereas substantially no accumulation of neutrophils is observed.

The following Examples illustrate the present invention.

EXAMPLE 1

Production and Testing of Bronchoalveolar Lavage Fluid

METHODS: Male Dunkin Hartley guinea-pigs (300-400 g) were sensitisedwith intraperitoneal ovalbumin (OA, 1 mg) on day 1 followed by exposureto aerosolised antigen (2% OA for 5 min using an ultrasonic nebuliser)on day 8 (6). On day 15-21, animals were pretreated with anantihistamine to prevent acute fatality (pyrilamine, 10 mg kg⁻¹, i.p.)and challenged by exposure to aerosolised antigen (1% OA for 20 min). Atdifferent times after antigen challenge animals were treated withatropine (0.06 mg kg⁻¹, i.p.) to prevent broncho-constriction and killedwith a barbiturate overdose. Broncho-alveolar lavage was performed with4 ml saline. Samples were centrifuged to remove cells and thesupernatant stored at -20° C. prior to assay. BAL samples were assayedby intradermal injection (0.1 ml) into guinea-pigs previously givenintravenous injections of 5×10⁶ 111 In-eosinophils (elicited in donoranimals by repeated intraperitoneal injections of horse serum andpurified on discontinuous Percoll gradients, >94% purity) (11,12). After4 hours, assay animals were killed and the skin sites punched out forgamma-counting. Data are presented as the mean±s.e.m. and were tested byone way analysis of variance. A p value of <0.05 was consideredstatistically significant.

RESULTS: FIG. 1 shows, schematically, procedures for the generation andmeasurement of eosinophil chemoattractant activity in vivo. FIG. 2illustrates the time course of generation of eosinophil chemoattractantactivity in lungs of sensitised guinea-pigs after antigen challenge(filled squares, n=4-10). Activity was measured in an in vivo skin assayof ¹¹¹ In-eosinophil accumulation in unsensitised guinea-pigs (3 testanimals per BAL sample). Significant activity was seen inbronchoalveolar lavage samples taken 0.5, 1.5, 3 and 6 h after antigenchallenge (compared to responses to intradermal saline, shown as thedotted line). No significant activity was observed in 24 h samples. Noactivity was seen in lavage samples obtained 3 h after sham (saline)challenge of sensitised animals (filled circles, n=5) or antigenchallenge of sham (saline) sensitised animals (filled diamonds, n=5).

EXAMPLE 2

Purification of Eotaxin from Bronchoalveolar Lavage Fluids

METHODS: Bronchoalveolar lavage (BAL) fluid collected from 25 sensitisedguinea-pigs (each lavaged with 4 ml, followed by 2×10 ml, saline) 3 hafter antigen challenge (1% OA, 5 min exposure) was applied to a cationexchange HPLC column (Ultropac TSK535CM, 7.5×150 mm). Activity waseluted at approx 1.4M ammonium acetate, pH5.5, and lyophilised for sizeexclusion HPLC (Ultropac TSK columns SWP, 7.5×75 mm, G4000SW, 7.5×600mm, and G2000SW, 7.5×600 mm, in series, equilibrated in 0.08% TFA).Activity eluted at approx 7-14 kDa. This was applied to a wide pore(300A) Vydac C18 reversed phase HPLC column (4×250 mm) in 0.08% TFA,eluted with a linear gradient of acetonitrile (0-80% ACN in 0.08% TFA,over 80 mins, at 1 ml min⁻¹) and 0.5 min fractions were collected.Aliquots (4%) of each fraction were lyophilised in the presence ofcarrier protein (BSA, <0.1 ng endotoxin mg⁻¹) and redissolved in 0.8 mlsaline for testing in the skin bioassays of ¹¹¹ In-eosinophil and ¹¹¹In-neutrophil accumulation over 2 hours (n=4). Eosinophils (99%, 0.5%neutrophils) were elicited with repeated intra-peritoneal injections ofhorse serum (11,12) and neutrophils (99.4%, 0.6% eosinophils) wereelicited with a single intraperitoneal injection of casein (5% w/v, 15ml) 17 h prior to purification on discontinuous Percoll gradients andlabelling with ¹¹¹ In.

RESULTS: The results are presented in FIGS. 3, 4 and 5. FIG. 3 shows thefinal reversed phase HPLC profile showing absorbance at 214 nm and theacetonitrile gradient. FIG. 4 shows that eosinophil chemoattractantactivity was seen in 2 peaks, corresponding to fractions 51+52 andfraction 54, which corresponded to discreet peaks of absorbance. FIG. 5shows that no significant neutrophil chemoattractant activity wasdetected in these fractions. In contrast, guinea-pig C5a des Arg (30%zymosan-activated plasma (11), approx 10 pmol/site) induced theaccumulation of both ¹¹¹ In-eosinophils (5211±893) and ¹¹¹In-neutrophils (9872±473). Fractions 50, 53, 55 and 56 consistently gavelittle or no activity in the guinea-pig skin bioassays of leukocyteaccumulation. No significant protein absorbance was detected in theremainder of the gradient (up to 80% acetonitrile).

EXAMPLE 3

Purity, Mass Analysis and Protein Sequence of Eotaxin

METHODS: 2% aliquots of each fraction were lyophilised, redissolved in10 μl SDS buffer, heated (95° C., 5 min) and 0.3 μl run on 8-25%gradient gels in a Pharmacia Phast System. Gels were visualised withsilver staining. Mass analysis was performed on fractions 51, 52 and 54using a Finnigan MAT Lasermat with α-cyano-4-hydroxycynnamic acid andsinapinic acid matrices. Mass measurements were calibrated internallyusing protein standards. 5% aliquots of each bioactive fraction (51, 52and 54) were applied directly to automated N-terminal sequence analysisusing fast cycles on an Applied Biosystems 477A containing amicrocartridge essentially as described (32). The amino-terminal 37, 35and 29 residues were obtained for fractions 51, 52 and 54 respectively.No differences between corresponding positions were found. The apparentinitial yields of these three analyses were all approximately 7-8 pmol.Thus fractions 51, 52 and 54 contained approx 200 pmol each, assuming70-80% sequencing yields. Gaps were found at positions 8,9 and 33,consistent with the presence of cysteine residues at these positions.Approximately 30 pmol of fraction 54 was reduced and alkylated bysequential treatment with 1 mM dithiothreitol for 5 min at 50° C. andthen 20 mm acrylamide for 30 min at 37° C. before digestion withalkylated trypsin (Promega) in 20 mM Tris/HCl, pH8.8, containing 0.5%Thesit. Peptides were separated using a Reliasil C18 (300 Å, 5 μm)column (1×150 mm) developed with a linear acetonitrile concentrationgradient in 0.08% tri-fluoroacetic acid at 50 μl/min on a Microm HPLCsystem. Purified peptides were subjected to N-terminal sequence analysisas above, but all four cysteine residues were positively identified asthe PTH-cys-S-β-propionamide derivative (33). Position 70 gave no PTHderivative in peptides T6 and T7 and is a probable position ofO-glyco-sylation.

RESULTS: The results are presented in FIGS. 6, 7, 8 and 9. FIG. 6 is aphotograph of the SDS-PAGE gel. For reference, human IL-8 (72 aminoacids, approx 8 kDa) was run in lanes A, B and C (12, 2.4 and 0.5 ng/0.3μl/lane respectively). Laser desorption time of flight mass analysisgave signals at 8.81 kDa (major) and 9.03 kDa (minor) for each offractions 51 and 52. Fraction 54 gave signals at 8.38 kDa (major) and8.15 kDa (minor).

FIG. 7 and SEQ.ID. NO. 1 show the amino acid sequence of eotaxin, whichwas determined by sequencing the intact molecule as well as peptidesderived from digestion with trypsin (T). N-terminal analyses showed thehighest homology with human MCP-1 (57%) and the tryptic peptides werereadily aligned by comparison with the human MCP-1 sequence. FIG. 8shows the amino acids sequence as confirmed by nucleic acid sequencing.FIG. 9 is a comparison of the eotaxin sequence with human MCP-1, MCP-2,MCP-3 (21), guinea-pig MCP-1 (24), human MIP-1α, MIP-2 and RANTES (18)showing conserved residues (shaded).

EXAMPLE 4

In vitro and in vivo Testing of Eotaxin

METHODS:

(i) Eotaxin was a pool of both peaks of bioactivity (fractions 51+52 andfraction 54) from the final reversed phase HPLC separation described inExample 2 (see FIGS. 3, 4 and 5). ¹¹¹ In-eosinophil accumulation inguinea-pig skin was measured over 4 h as described in Example 1 (seeFIGS. 1 and 2). In the same animals (n=4) additional sites were injectedwith eotaxin 30 minutes before killing.

(ii) For the binding studies, 4×10⁵ eosinophils were incubated with 0.1nM ¹²⁵ I-RANTES and various concentrations of cold ligand (50 μl at 0°C. for 2 h). The Hank's buffered salt solution contained 30 mM HEPES, 10mM EDTA, 0.1% sodium azide and 1% BSA at pH7.5. Results are the mean oftwo assays each done in triplicate.

(iii) For measurement of intracellular calcium levels human andguinea-pig eosinophils (10⁷ cells/ml in Ca²⁺ /Mg²⁺ -free PBS+0.1% BSA)were loaded with fura-2-acetoxymethyl ester (2.5 μM, 30min at 37° C.).After two washes cells were resuspended at 10⁶ cells/ml in Ca²⁺ /Mg²⁺-free PBS containing 10 mM HEPES, 0.25% BSA and 10 mM glucose (pH 7.4).Aliquots were dispensed into quartz cuvettes and the external [Ca²⁺ ]adjusted to 1 mM with CaCl₂. Changes in fluorescence were monitored at37° C. using a Perkin Elmer LS50 spectrophotometer at excitationwavelengths 340nm and 380nm and emission wavelength 510nm. [Ca²⁺ ]_(i)levels were calculated as described previously (34) using the ratio ofthe two fluorescence readings and a Kd for Ca²⁺ binding at 37° C. of224nM. Peripheral human eosinophils were prepared as describedpreviously (35) by density centrifugation on Percoll followed byimmunomagnetic removal of CD16⁺ neutrophils using the MACS system.Guinea-pig eosinophils were prepared as described in Example 1 (11,12).

(iv) To test for suppression of eposinophil accumulation in vivo usinghuman RANTES, accumulation of ¹¹¹ -labelled guinea-pig eosinophils inskin sites was measured as described above. Guinea-pigs were injectedwith 1.8 pmol eotaxin (n=2), 100 pmol RANTES (n=1) or both 1.8 pmoleotaxin and 100 pmol RANTES (n=2). Intradermal saline was used ascontrol.

(v) For testing of receptor antagonist activity, thefura-2-acetoxymethyl ester-loaded guinea-pig eosinophils were stimulatedwith 3 nM eotaxin with or without pretreatment with human RANTES.

(vi) To investigate the effect of aerosol exposure of guinea-pigs toeotaxin, naive guinea-pigs were exposed to an aerosol of either eotaxinor a control medium (n=8 per group). Exposure was performed by placingtwo animals in a single chamber and nebulising 24 pmol of eotaxindissolved in 10 ml PBS containing BSA carrier protein at 80 μg/ml over aperiod of 35-40 minutes. Control animals received aerosolised PBS/BSA inthe same manner. The animals were killed 20 hours after exposure toeotaxin or control medium. BAL fluid (5×10 ml HBSS, 10 mM EDTA, pH 7.35)was recovered and centrifuged (300 g, 20 min, 4° C.). Total BAL cellcount was determined by haemocytometer and differential cell countsperformed on stained (DiffQuick) cytospin preparations (3 per animal,400 cell counts per slide).

RESULTS: The results are presented in FIGS. 10, 11, 12, 13, 14, 15, 16,17 and 18.

FIG. 10 shows that guinea-pig eotaxin (1.6 pmol) induces significant ¹¹¹In-eosinophil accumulation in vivo 30 min (open squares, p<0.01) and 4 h(filled squares, p<0.01) after intradermal injection. In contrast, therecombinant human proteins, RANTES, MIP-1α and MCP-1, at doses up to 100pmol, were without effect over 4 hours. FIG. 11 inset shows that eotaxinand RANTES, but not the C-X-C chemokine IL-8, inhibit the binding of ¹²⁵I-RANTES (B₀ =14.4%) to guinea-pig eosinophils in vitro. FIG. 17 shows(a) that human RANTES does not induce significant eosinophilaccumulation and (b) that human RANTES inhibits to a substantial degreethe eosinophil accumulation induced by eotaxin, which suggests thatRANTES acts as a receptor antagonist for eotaxin in vivo.

FIG. 12 shows that eotaxin, RANTES and, at high concentration only,MCP-1 induce elevation of intracellular calcium levels in humaneosinophils in vitro. Traces are with eosinophils from one donor. In twoother donors 2 nM eotaxin gave a mean calcium elevation of 61 nM. In thethree donors (97.3±1.2% eosinophils) responses to 10 nM RANTES were194±74 nM [Ca²⁺ ]_(i) and responses to 100 nM MCP-1 were 93±38 nM [Ca²⁺]_(i). FIG. 13 shows that guinea-pig eotaxin, but not human RANTES orMCP-1, elevates intracellular calcium levels in guinea-pig eosinophilsin vitro. Traces are with cells from one donor. In three donors(97.5±0.8% eosinophils) responses were: 2 nM eotaxin, 90±13 nM [Ca²⁺]_(i) ; 100 nM RANTES, 2.0±1.7 nM [Ca²⁺ ]_(i) ; 100 nM MCP-1, 3.3±0.7 nM[Ca²⁺ ]_(i).

FIG. 14 and the inset FIG. 15 show that prior treatment of guinea-pigeosinophils with RANTES (100 nM) inhibits the increase in intracellularfree calcium levels seen when eosinophils are treated with eotaxin (3nM) alone. RANTES appears to be acting as a receptor antagonist inguinea-pig eosinophils. FIG. 16 is a dose-response curve using 3 nMeotaxin and increasing amounts of RANTES.

FIG. 18 shows that guinea-pig eotaxin, administered as an aerosol,induces eosinophil accumulation in guinea-pig airways in vivo, whereassubstantially no accumulation of neutrophils is observed.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 11                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 73 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Cavia por - #cellus                                   #Adult    (D) DEVELOPMENTAL STAGE:                                                      (F) TISSUE TYPE: Bronch - #oalveolar lavage fluid                   #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - His Pro Gly Ile Pro Ser Ala Cys Cys Phe Ar - #g Val Thr Asn Lys Lys         #                15                                                           - Ile Ser Phe Gln Arg Leu Lys Ser Tyr Lys Il - #e Ile Thr Ser Ser Lys         #            30                                                               - Cys Pro Gln Thr Ala Ile Val Phe Glu Ile Ly - #s Pro Asp Lys Met Ile         #        45                                                                   - Cys Ala Asp Pro Lys Xaa Xaa Trp Val Gln As - #p Ala Lys Lys Tyr Leu         #    60                                                                       - Asp Gln Ile Ser Gln Xaa Thr Lys Pro                                         #70                                                                           - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 73 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Cavia cob - #aya                                                (F) TISSUE TYPE: Bronch - #ial lavage fluid                         #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - His Pro Gly Ile Pro Ser Ala Cys Cys Phe Ar - #g Val Thr Asn Lys Lys         #                15                                                           - Ile Ser Phe Gln Arg Leu Lys Ser Tyr Lys Il - #e Ile Thr Ser Ser Lys         #            30                                                               - Cys Pro Gln Thr Ala Ile Val Phe Glu Ile Ly - #s Pro Asp Lys Met Ile         #        45                                                                   - Cys Ala Asp Pro Lys Lys Lys Trp Val Gln As - #p Ala Lys Lys Tyr Leu         #    60                                                                       - Asp Gln Ile Ser Gln Thr Thr Lys Pro                                         #70                                                                           - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base: N is inosine                           (B) LOCATION: 12                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base: N is inosine                           (B) LOCATION: 15                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base: N is inosine                           (B) LOCATION: 18                                                    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24CNAA CAAA                                                  - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: YES                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base: N is inosine                           (B) LOCATION: 10                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base: N is inosine                           (B) LOCATION: 16                                                    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                ATTT C                                                     - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 76 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cy - #s Cys Tyr Asn Phe Thr         #                15                                                           - Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Se - #r Tyr Arg Arg Ile Thr         #            30                                                               - Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Ph - #e Lys Thr Ile Val Ala         #        45                                                                   - Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Tr - #p Val Gln Asp Ser Met         #    60                                                                       - Asp His Leu Asp Lys Gln Thr Gln Thr Pro Ly - #s Thr                         #75                                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 74 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Ph - #e Asn Val Ile Asn Arg         #                15                                                           - Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Th - #r Arg Ile Thr Asn Ile         #            30                                                               - Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Th - #r Lys Arg Gly Lys Glu         #        45                                                                   - Val Cys Ala Asp Pro Lys Glu Arg Trp Val Ar - #g Asp Ser Met Lys His         #    60                                                                       - Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro                                     #70                                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 70 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Lys Ser Thr Thr Cys Cys Tyr Arg Phe Ile As - #n Lys Lys Ile Pro Lys         #                15                                                           - Gln Arg Leu Glu Ser Tyr Arg Arg Thr Thr Se - #r Ser His Cys Pro Arg         #            30                                                               - Glu Ala Val Ile Phe Lys Asp Lys Leu Asp Ly - #s Glu Ile Cys Ala Asp         #        45                                                                   - Pro Thr Gln Lys Trp Val Gln Asp Phe Met Ly - #s His Leu Asp Lys Lys         #    60                                                                       - Thr Gln Thr Pro Lys Leu                                                     #70                                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 71 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Gly Val Asn Thr Pro Thr Cys Cys Tyr Thr Ph - #e Asn Lys Gln Ile Pro         #                15                                                           - Leu Lys Arg Val Lys Gly Tyr Glu Arg Ile Th - #r Ser Ser Arg Cys Pro         #            30                                                               - Gln Glu Ala Val Ile Phe Arg Thr Leu Lys As - #n Lys Glu Val Cys Ala         #        45                                                                   - Asp Pro Thr Gln Lys Trp Val Gln Asp Tyr Il - #e Ala Lys Leu Asp Gln         #    60                                                                       - Arg Thr Gln Gln Lys Gln Asn                                                 #70                                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 69 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cy - #s Phe Ser Tyr Thr Ser         #                15                                                           - Arg Gln Ile Pro Gln Asn Phe Ile Ala Asp Ty - #r Phe Glu Thr Ser Ser         #            30                                                               - Gln Cys Ser Lys Pro Gly Val Ile Phe Leu Th - #r Lys Arg Ser Arg Gln         #        45                                                                   - Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gl - #n Lys Tyr Val Ser Asp         #    60                                                                       - Leu Glu Leu Ser Ala                                                         65                                                                            - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 68 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cy - #s Phe Ser Tyr Thr Ala         #                15                                                           - Arg Lys Leu Pro Arg Asn Phe Val Val Asp Ty - #r Tyr Glu Thr Ser Ser         #            30                                                               - Leu Cys Ser Gln Pro Ala Val Val Phe Gln Th - #r Lys Arg Ser Lys Gln         #        45                                                                   - Val Cys Ala Asp Pro Ser Glu Ser Trp Val Gl - #n Glu Tyr Val Tyr Asp         #    60                                                                       - Leu Glu Leu Asn                                                             65                                                                            - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 68 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cy - #s Phe Ala Tyr Ile Ala         #                15                                                           - Arg Pro Leu Pro Arg Ala His Ile Lys Glu Ty - #r Phe Tyr Thr Ser Gly         #            30                                                               - Lys Cys Ser Asn Pro Ala Val Val Phe Val Th - #r Arg Lys Asn Arg Gln         #        45                                                                   - Val Cys Ala Asn Pro Glu Lys Lys Trp Val Ar - #g Glu Tyr Ile Asn Ser         #    60                                                                       - Leu Glu Met Ser                                                             65                                                                            __________________________________________________________________________

We claim:
 1. An agent that inhibits or otherwise hinders the production,release or action of a chemoattractant protein which accomplishesattraction of eosinophils, induction of eosinophil accumulation, and/oractivation of eosinophils in vitro and in vivo, said chemoattractantprotein consisting of an amino acid sequence as set forth in SEQ ID NO:1or SEQ ID NO:2, said agent being an antibody which inhibits or otherwisehinders binding of said chemoattractant protein to a receptor.
 2. Acomposition comprising the agent of claim 1 and a pharmaceuticallyacceptable carrier.